The invention aims at using purine derivatives for the production of medicaments able to restore addressing of proteins from the endoplasmic reticulum to the plasma membrane. It aims in particular at treating mucoviscidosis.
Mucoviscidosis (CF) is the most common recessive autosomal lethal genetic disease in European and North American populations. The CF gene (locus 7q31) encodes a protein called Cystic Fibrosis Transmembrane Conductance Regulator (CFTR). Mutations of the CF gene cause abnormal transport of water and electrolytes through the cell membrane of various organs such as the lungs, sudoriparousglands, the intestine and the exocrine pancreas. Although there are over 1,000 mutations of the CFTR protein, the most common mutation (70% of patients) is the deletion of a phenylalanine in the NBF1 domain at position 508 (delF508). The main cause of mortality in CF patients is linked to this deletion and leads to infections or pulmonary insufficiency due to an increase in mucus viscosity. Such viscosity causes occlusion of respiratory airways and promotes infections by opportunistic bacteria. Furthermore, an aggravation is observed at the level of the digestive apparatus and the pancreas particularly (patient with pancreatic insufficiency). The CFTR protein is a glycoprotein of 1,480 amino acids, belonging to the ABC superfamily of membrane transporters. CFTR is a chloride ion channel localised in the apical plasma membrane of lung epithelial cells in healthy individuals. CFTR is responsible for trans-epithelial transport of water and electrolytes, thereby allowing hydration of lung airways in healthy individuals.
In CF patients homozygous for delF508 mutation, and more generally for class-II mutations (i.e. producing a protein that is absent from the cell membrane), CFTR is absent from the plasma membrane due to faulty addressing of this protein, which remains in the endoplasmic reticulum (ER). In such cases, hydration of lung airways is no longer functional. The delF508 deletion alters the folding of the NBF1 domain and prevents the full maturation of the protein, which is therefore degraded very early during biosynthesis. However, if the delF508 protein is able to reach the membrane, it works as a chloride ion channel.
One of the keys to treating this disease therefore consists in re-addressing delF508 to the plasma membrane of the cell, where the transport activity of delF508 can be stimulated by physiological agonists. Surprisingly, the inventors showed that derivatives known in particular for their anti-proliferative effect were capable of activating wild-type and mutated forms of CFTR, and inducing the relocation of delF508-CFTR protein to the plasma membrane, thereby restoring its transmembrane transport capacity. Generally speaking, such derivatives can restore a protein addressing error in cells. Furthermore, such derivatives have the advantage of being highly innocuous. The aim of the invention is therefore to provide a new use of these derivatives in order to produce medicaments for the treatment of mucoviscidosis and diseases related to a protein addressing error in cells.
The derivatives used according to the invention are purines substituted at positions 2, 6 and 9. Derivatives of this type have been described as kinase inhibitors. Such derivatives are in particular described in patent FR 95 14 237 and the patents and patent applications corresponding to PCT/FR96/01905, or in the article by Vesely et al, 1994, Eur. J. Biochem., 224, 771-786 II. The 2-(2-hydroxyethylamino)-6-benzylamino-9-methylpurine, commonly called olomoucine, is not within the scope of the invention.
The derivatives concerned are represented by formula I
where,
R2, R6 and R9, identical or different from each another, represent a halogen atom, an R—NH—, R—NH—NH—, NH2—R′—NH— or R—NH—R′—NH— group, wherein R is a straight or branched chain saturated or unsaturated alkyl group, an aryl or cycloalkyl group, or a heterocycle, and R′ is a straight or branched chain saturated or unsaturated alcoylene group, or an arylene or cycloalcoylene group, R and R′ each including 1 to 8 carbon atoms and being substituted, as the case may be, with one or more —OH, halogen, amino or alkyl groups,                R2 also possibly representing a heterocycle with, as the case may be, a straight or branched chain saturated or unsaturated alkyl group, an aryl or cycloaryl group, or a heterocycle, possibly substituted with one or more —OH, halogen, amino or alkyl groups,        R9 also possibly representing a straight or branched, saturated or unsaturated alkyl group, an aryl or cycloalkyl group,        R2 and R9 also possibly representing a hydrogen atom, with the exception of olomoucine.        
The invention aims in particular at using derivatives of formula I supra, where                R2 is chosen among the 3-hydroxypropylamino, 1-ethyl-2-hydroxyethylamino, 5-hydroxypentylamino, 1-D,L hydroxymethylpropylamino, aminoethylamino, 2-bis (hydroxyethyl)amino, 2-hydroxypropylamino, 2-hydroxyethylamino, chloro, R-hydroxymethyl-pyrrolidinyl, benzylaminohydroxyethyl, R,S-aminohydroxyhexyl, S amino-2-phenylhydroxyethyl, R amino-3-phenylhydroxypropyl, R,S aminohydroxypentyl, R aminohydroxypropyl, S aminohydroxypropyl, R(−)N-pyrrolidine hydroxymethyl groups,        R9 is chosen among methyl, isopropyl or cyclopentyl, and        R6 is chosen among benzylamino, 3-iodo-benzylamino, or isopentenylamino groups, when R9 represents an isopropyl group,        cyclohexylamino, cyclomethylamino, or 3-hydroxybenzylamino groups, when R9 represents a methyl group, and        a benzylamino group, when R9 represents a cyclopentyl group.        
These derivatives correspond to optical isomers and racemic mixtures, or as the case may be to geometric isomers. They more specifically involve 2-(R,S)(1-ethyl-2-hydroxyethylamino)-6-benzylamino-9-isopropylpurine or 2-(R)-(1-ethyl-2-hydroxyethylamino)-6-benzylamino-9-isopropylpurine, or even 2-(S)(1-ethyl-2-hydroxyethylamino)-6-benzylamino-9-isopropylpurine.
Said purine derivatives have the capacity to restore CFTR protein addressing to the plasma membranes of the cells and therefore constitute highly interesting compounds for the treatment of pathologies linked to such addressing errors. As illustrated by the various examples, they are particularly efficient for inducing the relocation of the delF508-CFTR protein to the membrane in mucoviscidosis, where this protein is retained in the endoplasmic reticulum, and thus restoring its trans-membrane transport capacity.
When developing medicaments, therapeutically efficient quantities of the active principles are mixed with pharmaceutically acceptable vehicles for the administration route chosen. Such vehicles may be solid or liquid. Thus, for oral administration, medicaments are prepared in the form of gelatine capsules, tablets, sugar coated tablets, capsules, pills, drops, syrups and similar. Such medicaments may contain 1 to 100 mg of active principle per unit. For parenteral administration (intravenous, subcutaneous, intramuscular injection), the medicaments are presented in the form of sterile or sterilisable solutions. They may also be in the form of suspensions or emulsions. The medicaments of the invention are more particularly administered in the form of aerosols.
Doses per administration unit may vary from 1 to 50 mg of active principle. The daily posology is chosen so as to obtain a final concentration not exceeding 100 μM of purine derivative in the blood of patients under treatment. Other characteristics and advantages of the invention will be described in the results reported below in order to illustrate the invention.